13C NMR Evidence for Pyruvate Kinase Flux Attenuation Underlying Suppressed Acid Formation in Bacillus subtilis

2000 ◽  
Vol 16 (2) ◽  
pp. 169-175 ◽  
Author(s):  
C. Phalakornkule ◽  
B. Fry ◽  
T. Zhu ◽  
R. Kopesel ◽  
M.M. Ataai ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Kinase ◽  
13C Nmr ◽  
Acid Formation

scholarly journals Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

2000 ◽  
Vol 66 (9) ◽  
pp. 4045-4049 ◽  
Author(s):  
B. Fry ◽  
T. Zhu ◽  
M. M. Domach ◽  
R. R. Koepsel ◽  
C. Phalakornkule ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Kinase ◽  
Acid Production ◽  
Metabolic Flux ◽  
Substantial Reduction ◽  
Acid Formation ◽  
High Concentration ◽  
Activity Measurements ◽  
High Concentrations

ABSTRACT Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pykmutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of thepyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pykmutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Pyruvate kinase of Bacillus subtilis

1972 ◽  
Vol 268 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Martin Diesterhaft ◽  
Ernst Freese
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Kinase

Enhanced recombinant protein production in pyruvate kinase mutant of Bacillus subtilis

2009 ◽  
Vol 85 (6) ◽  
pp. 1769-1778 ◽  
Author(s):  
Zhiwei Pan ◽  
Drew S. Cunningham ◽  
Tao Zhu ◽  
Kaimin Ye ◽  
Richard R. Koepsel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Pyruvate Kinase ◽  
Recombinant Protein Production ◽  
Protein Production

scholarly journals Engineering of Bacillus subtilis for Enhanced Total Synthesis of Folic Acid

2005 ◽  
Vol 71 (11) ◽  
pp. 7122-7129 ◽  
Author(s):  
T. Zhu ◽  
Z. Pan ◽  
N. Domagalski ◽  
R. Koepsel ◽  
M. M. Ataai ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Folic Acid ◽  
Total Synthesis ◽  
Pyruvate Kinase ◽  
Flux Analysis ◽  
Single Mutation ◽  
Acid Yield ◽  
Mutation Strategies ◽  
B Vitamin ◽  
Acid Precursor

ABSTRACT We investigated whether the yield of the B vitamin folic acid could be elevated in Bacillus subtilis. Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the Escherichia coli aroH gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor, para-aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed aroH, and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent B. subtilis 168 strain.

Regulating Expression of Pyruvate Kinase in Bacillus subtilis for Control of Growth Rate and Formation of Acidic Byproducts

2008 ◽  
Vol 22 (5) ◽  
pp. 1451-1455 ◽  
Author(s):  
Zhiwei Pan ◽  
Tao Zhu ◽  
Nathan Domagalski ◽  
Saleem Khan ◽  
Richard R. Koepsel ◽  
...  
Keyword(s):  
Growth Rate ◽  
Bacillus Subtilis ◽  
Pyruvate Kinase

scholarly journals Potensi Antibakteri Ekstrak Metanol dan Senyawa Aromatik dari Buah Wualae (Etlingera elatior)

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Sahidin Sahidin ◽  
Syefira Salsabila ◽  
Wahyuni Wahyuni ◽  
Fristiohady Adryan ◽  
Imran Imran
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Bacillus Subtilis ◽  
Streptococcus Mutans ◽  
13C Nmr ◽  
Vanillic Acid ◽  
Aromatic Compounds ◽  
Salmonella Enterica ◽  
Etlingera Elatior

Buah tanaman wualae (bahasa Tolaki) atau Etlingera elatior digunakan oleh masyarakat Tolaki (Sulawesi Tenggara) sebagai bumbu masak dan obat tradisional. Untuk mengungkap potensi tersebut, khususnya buah wualae, dilakukan kajian fitokimia dan farmakologi buah wualae. Kajian fitokimia meliputi isolasi dan penentuan struktur dikerjakan dengan metode kromatografi (kromatografi vakum cair dan kromatografi radial) dan spektroskopi (1H dan 13C NMR). Kajian farmakologi meliputi uji aktivitas biologi terhadap enam jenis bakteri (Bacillus subtilis FNCC 0060, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923, dan Streptococcus mutans ATCC 25175). Hasilnya menunjukkan tiga senyawa fenolik telah berhasil diisolasi dan diidentifikasi dari buah wualae yaitu asam vanilat (1), asam p-hidroksibenzoat (2), dan asam kumarat (3). Ketiga senyawa tersebut pertama kali dilaporkan berhasil diisolasi dari buah E. elatior. Aktivitas antibakteri ekstrak metanol buah wualae dan tiga senyawa hasil isolasi menunjukkan bahwa sampel tersebut paling aktif terhadap bakteri S. mutans. Dengan demikian aktivitas senyawa-senyawa hasil isolasi dari buah E. elatior  berkorelasi dengan aktivitas ekstrak metanol buah E. elatior  terhadap S. mutans, dimana senyawa paling aktif adalah asam vanilat. Ekstrak yang dominan mengandung asam vanilat dari tanaman ini dapat dikembangkan sebagai herbal antibakteri S. mutans seperti herbal karies gigi dan obat kumur. Kata kunci: Etlingera elatior, wualae, buah, senyawa aromatik, antibakteri. The fruits of wualae (Tolakinese) or Etlingera elatior is utilized by Tolakinese (Sulawesi Tenggara) as spices and traditional medicine. To reveal the potencies, a study on phytochemical and pharmacological aspects have been carried out. The phytochemical study includes isolation and structure determination used chromatography methods (vacuum liquid chromatography and radial chromatography) and spectroscopy methods (1H NMR and 13C NMR). Biological activity was evaluated against some bacteria that are Bacillus subtilis FNCC 0060, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923, and Streptococcus mutans ATCC 25175). The results showed that three aromatic compounds had been isolated and identified that are vanillic acid (1), p-hydroxybenzoic acid (2) and coumaric acid (3). All compounds are firstly reported that successfully isolated from fruits of Wualae. Antibacterial potency indicated that the methanol extracts and the isolated compounds are the most active towards S. mutans and have good activity correlation between the extracts and the compounds. Vanillic acid is the most active compound from E. elatior fruits. Thus, the extracts contain dominantly vanillic acid from          E. elatior fruits can be developed to be antibacterial herbals caused by S. mutans. Keywords: Etlingera elatior, wualae, fruits, aromatic compounds, antibacterial activity.

scholarly journals The Absence of Pyruvate Kinase Affects Glucose-Dependent Carbon Catabolite Repression in Bacillus subtilis

Metabolites ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 216 ◽  
Author(s):  
Sousa ◽  
Westhoff ◽  
Methling ◽  
Lalk
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Kinase ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Carbon Sources ◽  
Defined Medium ◽  
Central Carbon Metabolism ◽  
Alternative Pathways ◽  
Distribution Point ◽  
Nutritional Compounds

Pyruvate is a key intermediate of diverse metabolic pathways of central carbon metabolism. In addition to being the end product of glycolysis, pyruvate is an essential carbon distribution point to oxidative metabolism, amino acid and fatty acid syntheses, and overflow metabolite production. Hence, a tight regulation of pyruvate kinase (Pyk) activity is of great importance. This study aimed to analyze targeted metabolites from several pathways and possible changes in Bacillus subtilis lacking Pyk. Wild type and Δpyk cells were cultivated in chemically defined medium with glucose and pyruvate as carbon sources, and the extracted metabolites were analyzed by 1H-NMR, GC-MS, HPLC-MS, and LC-MS/MS. The results showed that the perturbation created in the pyruvate node drove an adaptation to new conditions by altering the nutritional compounds’ consumption. In Δpyk, pyruvate, which is subject to glucose-dependent carbon catabolite repression, did not comply with the hierarchy in carbon source utilization. Other metabolic alterations were observed such as the higher secretion of the overflow metabolites acetoin and 2,3-butanediol by Δpyk. Our results help to elucidate the regulatory transport of glucose and pyruvate in B. subtilis and possible metabolic reroute to alternative pathways in the absence of Pyk.

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